Autism and related diseases, collectively known as autism spectrum disorders (ASD), are the fastest growing neurodevelopmental disorders in the U.S., affecting as many as one in 200 children. Very little is known about the causes of ASD, which appear to include both genetic and environmental factors. We propose to use induced pluripotent stem cell technology to compare the development of autistic, Fragile X (mental retardation), and normal neuronal cells in vitro in an effort to understand the underlying pathology that leads to the neurological dysfunction. We propose to generate iPSCs from affected individuals and controls, characterize them using high-throughput genetic and epigenetic methods for comparison with our large existing database of pluripotent and neural stem cell molecular profiles, and study their differentiation in vitro using a set of molecular and physiological tools. For the first (R21) phase, we will focus on discovery and the development of tools. We will obtain autistic fibroblasts from Dr. Philip Schwartz, a longtime collaborator who has recently launched an NIH- sponsored program to generate autism fibroblasts and iPSCs as a resource for the research community. We will perform pilot studies with a limited number of patient-specific lines, providing Dr. Schwartz with the iPSCs we generate using our established methods and new virus-free methods in development. We will follow their differentiation in vitro into a variety of neuronal cell types, characterizing selected subpopulations for global expression of mRNA and microRNAs, DNA methylation (epigenetic) profile, and copy number variation (SNP genotyping). Finally, we will systematically compare the properties of differentiated disease-specific and control iPSC-derived neurons using optical and electrophysiological methods. For the second (R33) phase, we will incorporate more cell lines (produced by our group and by Dr. Schwartz) and optimize the tools that are most promising. First we will expand the Autism Stem Cell Matrix database of molecular profiles and use bioinformatics tools to identify significant differences between disease-specific and control cultures. We will extend the functional studies by maturing the cells in vitro into three-dimensional cortex-like aggregates and characterizing their synaptic interactions in this model of early CNS development. Finally, we will combine a lineage tracing tool (Brainbow) with cell type-specific gene expression methods to focus on molecular differences among neuronal subtypes. Our goal is to produce a comprehensive analysis comparing functional development of autistic, Fragile X, and normal neuronal cells in vitro. From this detailed study, we hope to provide clues to the developmental causes of autism. PUBLIC HEALTH RELEVANCE: Autism is a disorder of neural development whose causes are virtually unknown; largely because of this lack of knowledge, there are no consistently successful treatments for this disease that is expected to affect 4 million children and their families in the US by the end of the next decade. The goal of this project is to shed light on the causes of autism by studying development of the nervous system in a controlled tissue culture environment using sophisticated molecular tools. We will obtain cells from the skin of autistic patients, using a virtually painless procedure, and turn them into induced pluripotent cells (iPSCs) that can develop into every cell type in the body. We will study the development of these cells into nerve cells in tissue culture, comparing them with cells from unaffected children. From this detailed study, we hope to learn what goes wrong during brain development in autistic children, and help develop a strategy for effective treatment.